Detection of Hepatitis B virus DNA polymerase gene mutations

HBV is an important health problem affecting one third of the world population and more than 350 million people being carriers. Chronic B hepatitis is a serious disease which must be treated because of its potential to progress to cirrhosis and hepatocellular carcinoma. Interferon and nucleoside analogs are used for treatment. Among the nucleoside analogs, lamivudine (LAM) is frequently the drug of choice because of its high efficacy, good tolerability, low rate of adverse effects and low cost compared with other drugs. Mutations that arise during LAM treatment and cause resistance are usually seen at the 6th-8th months of treatment and the frequency increases with the duration of treatment. LAM resistance should be determined in order to monitor treatment success and to continue treatment with alternative drugs.Monitoring and reporting of changes in HBV pol gene during treatment will help to define the new drug resistance patterns and clinical consequences. Based on these data, this study aimed to1.Determine the incidence and timing of development of LAM resistance mutations,2.Compare sequence analysis, restriction enzyme analysis (REA) and strip hybridization (LiPA) in determining YMDD motif mutations in HBV pol gene,3.Determine the relationship between the timing of mutation appearance and rise in viral load / ALT level in cases with LAM resistance mutation,4.Examine S mutations in regions that overlap with LAM resistance mutations,5.Determine other sequence changes that develop in HBV DNA pol gene,6.Determine the viral genotype/subtypes of the cases.The study group has been composed of chronic B hepatitis cases who were followed by the Dokuz Eylül University Medical Faculty, Division of Gastroenterology and in whom lamivudine treatment was started. The study population was divided into two subgroups:·Group A (n=30): Cases who were followed-up for at least one year and of whom serial serum samples from the pretreatment period and of 3rd, 6th, 9th and 12th months of treatment were available.·Group B (n=16): Cases who did not have serial serum samples but who could provide samples from time periods of the treatment. In this group, eleven of the cases had a sample from the 12th month, 14 cases had a sample from the 24th month and 9 cases had a sample from the 36th month of treatment.Emergence ratio of YMDD mutants in Group A was evaluated according to the years by sequencing. Number of cases who have YMDD mutants was 6/30 (20%) in first year, was 8/20 (40%) in second year and was 4/10 (40%) in third year. For all cases of Group A and Group B, ratio of cases with YMDD mutant determined by sequencing and LiPA method was 23.1% (9/39 cases) end of the 1st year of lamivudine therapy, was 36.4% (12/33 cases) at the end of the 2nd year and was 35.0% (7/20 cases) at the end of the 3rd year.The results of sequencing analysis of 123 serum samples obtained from followed-up cases who treated with lamivudine at different stage of therapy (before, during and after therapy) were compared with the results of LiPA and REA methods. LiPA test was more successful than sequencing and REA for the determination of variants. LiPA test was more sensitive to determine of minor viral populations for codon 204 than sequencing analysis by 3.25% for 4/123 samples. It was observed the LiPA test was more sensitive than REA for 64/123 samples at codon 180 by 52% and for 63/123 samples at codon 204 by 51.2%. Results of sequencing and LiPA were in consistence by 99.9% for codon 180 (122/123 samples) and by 94.3% for codon 204 (116/123 samples). Although LiPA test is usually more successful to detect the minor populations , it could not detect one of the mixed viral populations in two samples (one for codon 180, one for codon 204). While for two samples of a patient, rt M204I mutation was detected by sequencing, hybridization bands for YMDD and mutant sequence specific probes were not observed by LiPA.REA method could not give any results below 103 copy/mL HBV-DNA level and any results were obtained for 62 samples (50.4%). Sequencing method was more sensitive to detect samples containing mixed viral populations than REA for codon 180 by 52.0% (64/123 samples), for codon 204 by 52.8 (65/123 örnek). In these samples either any results were obtained with REA method because of viral load or could not be detected minor types in mixed viral populations. REA method was more successful than sequencing to identify mixed viral populations for only one sample at codon 204 and than LiPA for one sample at codon 180 and one sample at codon 204.In this study while YMDD mutant was detected in 50% of the patient group, the viral resistance was observed for 80% of cases (12/15 cases) have YMDD mutant. In cases with YMDD mutants, mutations were detected at the 18.8th month. Mutations were detected at least 3rd month of therapy (one case). Significant increase was observed for viral load at average 5 months later than detected YMDD mutations. Generally ALT elevations were occurred after an increase of viral load and were detected after at average 26th months of therapy.YMDD mutant was detected for only one case in Group A (n=30) with sequencing and LiPA methods before LAM treatment. rtM204I mutation was constituted the minor population and together with dominant YMDD type. YIDD mutant observed before therapy disappeared with starting therapy and again was detected with sequencing and LiPA methods at the 30th months of therapy. Existence of viral populations with YMDD and YIDD sequences detected by sequencing and LiPA was confirmed by clonal analysis for pretreatment sample. It was observed that all seven clones obtained from clonal analysis of the serum sample at 9 th months of therapy had YMDD sequences.The first observation YMDD mutations were YIDD (6 cases), YMDD+YIDD (4 cases), YMDD+YIDD+YVDD (3 cases), YIDD+YVDD (1 case) in study group A cases during LAM therapy. In 5 cases who developed a mutation, we were capable of determining a pattern of consecutive mutations. In these cases first exchange YMDD viruses with YIDD mutants and after a while a shift from YIDD mutants to YVDD mutants are observed. In cases compensatory rtL180M mutation was observed at the same time with YVDD/YIDD mutation or later. 13 of rtL80I/V mutations detected at 16 samples in 12 cases has YIDD mutation and 3 of mentioned samples has YIDD+YVDD mutation.In cases who developed LAM resistance, W196S/L, I195M, E164D mutations in the S gene produced as a consequence of the rt M204I, rtM204V, rtV173L mutations in the polymerase gene.N76D (3 cases), R110G (2 olgu), L115Y, S117Y, N123D, G127R, T128N (2 cases), T128I, N139K, L144C, L145M, T184I, V214E, S219A, F221Y, T225N L229W, L231V, P237H, N238H, Y245H, K270R sequence changes detected by sequencing were observed in the pol gene of cases with LAM resistance mutations.In serum samples of 30 cases, HBV genotypes were evaluated using by sequencing. As a result of phylogenetic analysis, sequences of all cases was observed that clustered with genotype D sequences. It was determined, 7 of cases infected with ayw3 subtype (23.3%) and 23 of cases infected with ayw2 subtype (76.7%).